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Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen ( actin A3 ,  actin A1 ,  GAPDH ,  G3PDH ,  E2F ,  rp49 ) in four lepidopteran insects  Bombyx mori  L. (Lepidoptera: Bombycidae),  Plutella xylostella  L. (Plutellidae),  Chilo suppressalis  Walker (Crambidae), and  Spodoptera exigua  Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ΔCt analysis were used to evaluate these HKGs. Across different developmental stages,  actin A1  was the most stable in  P. xylostella  and  C. suppressalis , but it was the least stable in  B. mori  and  S. exigua. Rp49  and  GAPDH  were the most stable in  B. mori  and  S. exigua , respectively. In different tissues,  GAPDH ,  E2F , and  Rp49  were the most stable in  B. mori ,  S. exigua , and  C. suppressalis , respectively. The relative abundances of  Siwi  genes estimated by 2 -ΔΔCt  method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR.

Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects